Strain bifidobacterium lactis 676 useful in preparation of fermented-milk products, healthy products, fermented and non-fermented foodstuffs, bioactive supplements, probiotics, and cosmetic agents

FIELD: biotechnology, food processing industry, medicine, veterinary.

SUBSTANCE: strain Bifidobacterium lactis 676 is isolated from bulk of healthful infant and is characterized with increased biological activity, environmental strength, accelerated propagation on artificial cultural media, acid-forming and antagonistic activity against pathogenic and opportunistic microorganisms. Strain is useful in preparation of functional foodstuff, bioactive supplement, cosmetic and toiletry agents.

EFFECT: bifidus-bacterium strain for manufacturing of products with excellent probiotic and healthy effect for human and animals.

2 tbl, 6 ex

 

The technical field

The invention relates to new strains of bifidobacteria and their use in food, cosmetic, biotechnology, medical industry and animal health to strengthen health. The inventive strain, in particular, can be used in the preparation of fermented milk, fermented and non-fermented food starters, cosmetics, dietary supplements and bacterial preparations.

The level of technology

Due to unfavorable environmental conditions, long and sometimes non-systemic antibiotic therapy increases the number of people and animals suffering from disorders of the normal microflora. For the treatment and prevention of these disorders is gaining attention as drugs, with probiotic action, and food products containing bifidobacteria.

Products containing bifidobacteria, are effective in the prevention and treatment of various gastrointestinal diseases, and intestinal dysbacteriosis caused intestinal and other diseases, prolonged antibiotic therapy, radiotherapy and chemotherapy, stress effects, adverse environmental conditions and other factors.

Bacterial resistance to the conditions of gelado the but-intestinal tract is an important criterion for the selection of strains health promoting.

The quality of therapeutic products and medicines is largely due to the properties of the producer strain - specific activity and adaptability. A large range of biologically active and technologically advanced strains of bifidobacteria makes possible the development of new mono - and complex products of therapeutic nutrition and bakpreparatov, capable of providing high colonization resistance of the intestines of humans and animals, to restore the normal microflora and reduce the incidence of various intestinal infections.

Well-known foreign mono - and combination preparations - eubiotics and dairy products, including bifidobacteria species: Bifidobacterium bifidum (patent FR 2336886, 29.07.1977 And 23 With 9/12), Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis (patent FR 2411008, 06.07.1979, A 61 K 35/74), Bifidobacterium longum, Bifidobacterium breve (patent CH 637297, 29.07.1983, A 61 K 39/02), Bifidobacterium lactis (patent US 6379663, 14.03.2002, a 01 N 63/00.

In our country for the preparation of bacterial preparations and products for medical-diet used strains of bifidobacteria species: Bifidobacterium bifidum PMBC Ac-1578 (patent RU 2152993, 20.07.2000, C 12 N 01/20), Bifidobacterium bifidum PMBC S-1257 (patent RU 2108383, 10.04.1998, C 12 N 01/20), Bifidobacterium longum B379M, Bifidobacterium bifidum 791 (patent RU 2092064, 10.10.1997, A 23 C 09/12), Bifidobacterium adolescentis MC-42 (inventor's certificate SU 863639, 15.0.1981, C 12 N 15/00). In dairy products, enriched with bifidobacteria, use and foreign strains such as Bifidobacterium lactis HN-019 (patent RU 2208632, 20.07.2003, C 12 N 01/20), Bifidobacterium lactis 420 (specification on the strain, Bifidobacterium lactis 420 company "DANISCO", dated 02.05.2003).

Up to the present time nothing is known about the use of domestic strains of Bifidobacterium lactis in the above areas.

Closest to the claimed strain is Bifidobacterium lactis 420 (prototype) (specification on the strain, Bifidobacterium lactis 420 company "DANISCO", dated 02.05.2003). The strain has an antagonistic activity against some pathogenic and conditionally pathogenic microorganisms.

On a number of proven properties of known strain Bifidobacterium lactis 420 yields the claimed strain Bifidobacterium lactis 676, in particular it is more sensitive to phenol, bile and less active against some pathogenic microorganisms (see table 1 and table 2).

Disclosure of inventions

The present invention is to obtain a strain of microorganism of the species Bifidobacterium lactis for use in the manufacture of fermented milk products, fermented and non-fermented food starters, biologically active additives, bacterial preparations, cosmetics and other products containing bifidobacteria.

When the task is achieved following those who practical results; the strain has high biological activity, in particular high antioxidant activity (superoxidedismutase activity) in comparison with the information available in the literature, resistance to environmental conditions, multiplies rapidly on artificial nutrient media, has an acid-forming and antagonistic activity against pathogenic and conditionally pathogenic microflora. Obtaining strains with similar properties, contributes to the expansion of the range of probiotics used to maintain health, in particular for normalization of microflora in the gastro-intestinal and genitourinary tracts, mucous membranes of the skin of humans and animals.

The task is implemented by using Bifidobacterium lactis 676 (Ac-1678 PMBC) in the composition of starter cultures, food, bacterial, cosmetics, etc.

Strain Bifidobacterium lactis 676 can be used in mono or composite probiotic concept or production of various cultures, fermented milk, fermented and non-fermented foods, dietary supplements, bacterial preparations, cosmetic preparations.

Strain Bifidobacterium lactis 676 selected from the contents of the intestine of a healthy child and deposited in PMBC (Russia, Moscow, 113545, 1st Road travel, 1 GNIH-PMBC) # AU-1678.

The inventive strain survives in vitro conditions that mimic the conditions of the gastrointestinal tract (GIT), in particular, in the presence of 40% bile, 0.4% phenol, digestive enzymes and low pH values.

The inventive strain can be used in the production of dairy products, fermented foods, such as yeast and the like, non-fermented foods, such as juices, drinks, etc., sourdough and biologically active additives to food. Also the strain Bifidobacterium lactis 676 can be widely used for cosmetics, such as creams, lotions, nourishing mask, face lotion, etc.

In addition, the strain of Bifidobacterium lactis 676 may be used in the production of bacterial preparations in the form of candles, oral, and other tools.

Cultural, morphological and physiological-biochemical properties of the strain.

Cells are gram-positive, fixed, nesporoobrazuth, catalytically, anaerobic, polymorphic sticks with bifurcation or thickening at 1-2 ends; in a liquid nutrient medium to grow over the entire height of the volume, except for the zone of aerobiosis; growth on agar medium MMC under anaerobic conditions to form small (up to 1 mm in diameter) round colorless colonies.

The optimum temperature for growing 37-39°C.

Strain sbraga the AET glucose, lactose, sucrose, maltose, salicin, xylose, arabinose, raffinose and mannose with the formation of acetic and lactic acids, without the formation of gas;

skachivat milk for 30 h with the formation of a clot;

actively increasing microbial mass and growth in corn-lactose medium (MMC-1) (MMC-1: corn-milk formula - 15 g; peptone and 15 g; lactose - 9 g; ascorbic acid, 0.5 g; sodium citrate trehzameshchenny - 0.6 g; magnesium sulfate - 0.12 g; potassium phosphate one-deputizing 2 g; sodium phosphate disubstituted - 1 g; agar, 3 g; distilled water to 1 l), bifidum environment (BF) (composition bifidum environment: glucose and 7.5 g; lactose - 2.5 g; pancreatic hydrolysate of casein - 30 g; sodium chloride, and 2.5 g; magnesium sulfate, 0.5 g; L-cysteine, 0.5 g; ascorbic acid, 0.5 g; yeast extract 5 g; sodium acetate to 0.3; agar - 0.75 g; distilled water to 1 l);

maximum accrue microbial mass to 12-24 hours, reaching in 1 ml Ig CFU (colony forming unit)equal 8-9;

exhibits antagonistic properties against pathogenic and conditionally pathogenic microflora (table 1 and 2 shows at the end of the description), namely: more than 10 times less Klebsiella pneumoniae, more than 100 times reduced the amount of Candida albicans and Proteus mirabilis and more than 1000 times less Proteus vulgaris, Shigella sonnei, Citrobacter freundii, Bacillus subtilis compatible with the om cultivation in vitro compared to control.

Thus, the proposed strain Bifidobacterium lactis 676 tech as actively propagated in a nutrient medium with the accumulation of biomass production in short-term (12-24 h) timing of cultivation with a high concentration of microbial cells (Ig CFU˜9), has the acid and any abscopal properties against pathogenic and conditionally pathogenic microflora, high antioxidant activity (superoxidedismutase activity, SOD activity equal 53,9±0.1 u/mg protein).

In addition, the study of the spectrum of sensitivity to antibiotics and antibacterial drugs revealed that the strain Bifidobacterium lactis 676 has a natural resistance to aztreonam, polymyxin, streptomycin, sulfanilamide, tobramycin, fusidin and sensitive to chloramphenicol, vancomycin, lincomycin, Meropenem, oxacillin, piperacillin, rifampicin, furagin, furadonin, furazolidone, cefepime, Cefotaxime, Ceftriaxone, cefuroxime, erythromycin, ceftazidime.

Thus, for a number of properties and total strain Bifidobacterium lactis 676 possess greater biological activity in comparison with the known strains of Bifidobacterium lactis.

Strain Bifidobacterium lactis 676 resistant to phenol and bile; resistance to low pH and digestive enzymes; expressed antagonistic activity against Proteus vulgaris,Proteus mirabilis, Klebsiella pneumoniae, Shigella sonnei, Citrobacter freundii, Bacillus subtilis; natural resistance to aztreonam, polymyxin, streptomycin, sulfanilamide, tobramycin, fusidin; good adaptability (the ability of the strain to grow in a big titer equal to Ig KOE˜9, in selected environments and to be well preserved in fermented milk products during the required extended shelf life); a pronounced antioxidant activity.

The ability of strain Century lactis 676 grow into a big title in a nutrient medium in a short period of cultivation, his antagonistic, acid, antioxidant activity, the ability to persist for long periods of time in milk products, the ability to synthesize some vitamins and other useful properties make possible the use of the strain in the composition of various food products and means of therapeutic and prophylactic purposes.

The implementation of the invention.

Example 1. Using Bifidobacterium lactis 676 in the composition of dietary supplements.

1 passage: dried Bifidobacterium lactis 676 in the amount of ˜0.1 g of dry biomass contribute in tubes with nutrient medium and titrated by the method of ten-fold dilutions up to 10-9. Incubated at a temperature of 37-38°before the formation of visible growth in the last dilutions.

2 passage: grown cool the tour from 10 -7-9dilutions up to 10% contribute to the nutrient medium and cultured at a temperature of 37-38°C for 12-24 h before formation of the dense growth of the visible area of aerobiosis.

3 passage: the biomass in an amount up to 10% of the volume contribute to the accumulation and cultivate 12-24 h at 37-38°to obtain a biomass content of live Bifidobacterium lactis 676 not less than 108CFU/ml

The result is biologically active additive, which is a suspension of cells of Bifidobacterium Bifidobacterium lactis 676 with a titer of not less than 108CFU/ml, if necessary (using concentration) can be obtained concentrate cells of bifidobacteria with a higher titer.

Example 2. Using Bifidobacterium lactis 676 for the preparation of non-fermented foods.

1 passage: dried Bifidobacterium lactis 676 in the amount of ˜0.1 g of dry biomass contribute in tubes with nutrient medium and titrated by the method of ten-fold dilutions up to 10-9. Incubated at 37-38°before the formation of visible growth in the last dilutions.

2 passage: a grown culture of 10-7-9dilutions up to 10% contribute to the nutrient medium and cultured at a temperature of 37-38°C for 12-24 h before formation of dense growth with a highly visible area of aerobiosis.

3 passage: the biomass in an amount up to 10% by volume is introduced into the environment, accumulation and cultivate 12-24 h at 37-38° To obtain the biomass content of live bifidobacteria not less than 108CFU/ml

Obtaining non-fermented food: growing up and, if necessary, concentrated biomass Bifidobacterium lactis 676 bring in the food product (milk, juices, etc), mix and pour into containers. The result is a product with a concentration of live bifidobacteria not less than 106CFU/ml

Example 3. Preparation of fermented milk product of therapeutic and prophylactic purposes using the proposed Bifidobacterium lactis 676 as a component of starter cultures.

1 passage: lyophilized strain introduced into the environment of cultivation and titrated by the method of ten-fold dilutions up to 10-9. Incubated at 37-38°before the formation of visible growth in the last dilutions.

2 passage: a grown culture of strain 676 of the last dilution contribute to the accumulation of up to 10% and cultured at a temperature of 37-38°to obtain a biomass containing bifidobacteria not less than 108CFU/ml

3 passage: the biomass in an amount up to 10% of the volume contribute to the accumulation and cultivate 12-24 h at 37-38°to obtain a biomass content of live bifidobacteria not less than 108CFU/ml

Receiving fermented milk product: the biomass of the strain 676 in the amount of up to 10% make Paster is transferring milk mixture at a temperature of 37-38° Together with other starter cultures used for the norm, to cultivate the formation of a clot (pH 4.5 to 4.6). The product with the concentration of live bifidobacteria not less than 106CFU/ml cooled to 22°C, poured into containers and Doklady to 6°C. the Number of passages may be increased depending on the volume of production.

Example 4. Using Bifidobacterium lactis 676 to obtain bacterial drugs.

The culture of the strain of the 1st or 2nd passage (example 1) contribute to the nutrient medium, cultured at a temperature of 37-38°to obtain the biomass of bifidobacteria with the contents of living cells is not less than 108CFU/ml To the obtained biomass add a protective environment drying (for example, Saharsa-gelatin medium, skim milk). The resulting suspension is poured into ampoules, vials and other containers, frozen at minus 40-45°and lyophilizers from the frozen state in a vacuum drying apparatus. The resulting preparation contains not less than 108living cells of bifidobacteria in 1, Forming a dose adequate level of consumption of probiotic microorganisms per day, equal to not less than 5×108-5×1010SOME (bifidobacteria), you can use the drug as a drug of probiotic.

Example 5. Using Bifidobacterium lactis 676 for gaining the cosmetics.

The culture of the strain of the 1st or 2nd passage (example 1) contribute to the nutrient medium, cultured at a temperature of 37-38°to obtain the biomass of bifidobacteria with the contents of living cells is not less than 108CFU/ml

Getting cosmetics: grown biomass (if necessary concentrate) Bifidobacterium lactis 676 enter into the composition of cosmetic products.

Example 6. The use of a strain of Bifidobacterium Bifidobacterium lactis 676 to obtain a fermented product.

The culture of the strain of the 1st or 2nd passage (example 1) contribute to the nutrient medium, cultured at a temperature of 37-38°to obtain the biomass of bifidobacteria with the contents of living cells is not less than 108CFU/ml

To obtain a fermented product type kvass grown biomass (if necessary concentrate) strain of Bifidobacterium Bifidobacterium lactis 676 contribute to the prepared base (water, sugar, honey, wort, ascorbic acid) together with Lactobacillus plantarum and Lactobacillus fermentum, mixed and cultured at 37°to obtain the beverage with acidity 65-85°So Then the drink is cooled and bottled. The result is ready therapeutic product containing 1 ml not less than 107live probiotic microorganisms.

PCs is mm Bifidobacterium lactis 676 PMBC Ac-1678, used to prepare dairy therapeutic products, fermented and non-fermented foods, dietary supplements, probiotic and cosmetics.



 

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